EP4116429A1 - Verfahren zur analyse einer probe zum nachweis einer aktiven form einer biologischen spezies - Google Patents

Verfahren zur analyse einer probe zum nachweis einer aktiven form einer biologischen spezies Download PDF

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Publication number
EP4116429A1
EP4116429A1 EP22182863.5A EP22182863A EP4116429A1 EP 4116429 A1 EP4116429 A1 EP 4116429A1 EP 22182863 A EP22182863 A EP 22182863A EP 4116429 A1 EP4116429 A1 EP 4116429A1
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EP
European Patent Office
Prior art keywords
sample
biological species
treatment
active
channel
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Pending
Application number
EP22182863.5A
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English (en)
French (fr)
Inventor
Anne-Gaëlle BOURDAT
Caroline DESVERGNE
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Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique CEA
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Publication of EP4116429A1 publication Critical patent/EP4116429A1/de
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

  • the present invention relates to a method for analyzing a sample to detect the presence therein of an active form of a biological species.
  • a known particular case is that of downy mildew which is a common disease present in agriculture and particularly destructive.
  • the pathogen responsible for this disease can be both dormant and active. For example, it is likely to develop on the leaves of plants and on the bunches of grapes. It is currently treated with repeated and systematic applications of fungicide, which lead to environmental pollution, toxicity and the development of more resistant strains.
  • biomolecular detection makes it possible to detect the presence of the biological species and possibly the quantity of biological species present, but not to know whether it is present in its dormant form or in its active form.
  • the system is particularly cumbersome to set up and requires continuous monitoring.
  • the method of the invention makes it possible to meet this need.
  • the active treatment of the second part of the sample comprises at least one chemical or biochemical treatment, carried out by flow injection of a substance that destroys the active form of the biological species into the second part of the sample. sample after concentration, in order to break its outer membrane and release its nucleic acids.
  • the active treatment is carried out by lysis of the biological species against a rough surface.
  • the heat treatment consists of heating by heating means of the second part of the sample to a given temperature and for a determined duration.
  • the optical treatment consists in exposing the second part of the sample to a light signal emitted at a given wavelength and for a determined duration.
  • the first concentration and the second concentration are implemented simultaneously in two identical microfluidic components in parallel.
  • the soft treatment and/or said active treatment are implemented in said micro-fluidic component, by flow injection through the first channel of the component.
  • the first quantitative detection and the second quantitative detection are carried out by biomolecular amplification.
  • the sample is obtained after a step of grinding and extracting a sample to be analyzed.
  • a targeted biological species may be present in the sample in a so-called dormant form (hereinafter F1) and in a so-called active form (hereinafter F2).
  • the dormant form F1 is the harmless and resistant form of the biological species while its active form F2 is an active and destructive form.
  • the terms "harmless” and “destructive” must of course be understood in relation with the environment in which the biological species is present, this environment being able to be a living being, a plant, a food matrix...
  • the detection principle used in the invention is also based on the fact that the biological species is often more fragile in its active form F2 than in its dormant form F1, in the sense that, when an active treatment is carried out on the sample, the DNA molecules of the active form F2 tend to break more easily than those of the dormant form F1.
  • the invention thus aims to propose a method making it possible to detect the presence of an active form F2 of a biological species in an ECH sample.
  • the ratio obtained between the quantity Q1 of biological species present in the first part A of the sample and the quantity Q2 of biological species present in the second part B of the sample it can then be determined whether the biological species is present and whether it is present in its active F2 and dormant F1 form, or only in its dormant F1 form.
  • a microfluidic component represented on the picture 3 .
  • This microfluidic component 1 comprises a housing comprising a bottom wall 10, a side wall 11 and an upper wall 12. All the walls of the housing will preferably be made of the same material. This material will in particular be capable of being able to undergo heating in a temperature range of between 20° C. and 100° C. Preferably, some walls of the case will be made of a transparent material. Preferably, the material used will be a plastic, for example of the PMMA (Poly(Methyl Methacrylate)) or COC (Cyclic Olefin Copolymer) type.
  • PMMA Poly(Methyl Methacrylate)
  • COC Cyclic Olefin Copolymer
  • Component 1 comprises a chamber 13 formed in the housing. This chamber represents the location in which both the purification/concentration and the detection of the target biological species can be carried out.
  • the chamber 13 is closed downwards by the bottom wall of the casing.
  • the micro-fluidic component includes a first channel 14 formed in the casing and arranged to inject fluids into the chamber or to evacuate fluids outside the chamber.
  • the first channel 14 comprises a first end comprising an opening formed for example through the upper wall 12 of the housing and a second end which opens into said chamber 13.
  • the first end of the first channel 14 is for example arranged vertically and its second end opens for example horizontally in the chamber 13.
  • the first end of the first channel is for example flared to apply the cone of a pipette to it or will be adapted to the type of device used to inject the fluid into the device.
  • it may be an opening having a “luer” type end piece to connect a syringe thereto or adapted to connect a fluidic circuit thereto.
  • the component comprises a second channel 15 made in the housing.
  • This second channel 15 also comprises a first end which communicates with the outside, forming an opening made for example through the upper wall of the casing and a second end which communicates with the space formed by the chamber 13.
  • This second channel 15 it is also possible to inject fluids into said chamber or to evacuate fluids outside of said chamber.
  • Its first end is for example arranged vertically and its second end horizontally.
  • the chamber 13 is placed between the first channel 14 and the second channel 15.
  • the first end of this second channel is for example flared to apply the cone of a pipette to it or will be adapted to the type of device used for inject the fluid into the device.
  • it may be an opening having a “luer” type end piece to connect a syringe thereto or adapted to connect a fluidic circuit thereto.
  • the chamber 13 can be closed by a membrane 18 which is advantageously flexible and stretchable, preferably transparent.
  • the upper wall 12 of the housing of the device thus comprises an opening which is hermetically covered by said membrane 18.
  • Said membrane is thus anchored in the housing by any suitable fastening solution, for example by gluing.
  • This membrane 18 will for example be composed of a film, for example a self-adhesive film of the PET type, of thickness, dimensions and constitution adapted to deform elastically, with respect to its anchoring points, in particular up to at the back of chamber 13.
  • transparent it is meant that the material used is at least partially transparent to visible light, to fluorescence or to luminescence, so as to allow at least 80% of this light to pass. It should thus be understood that it will be sufficiently transparent to see the interior of the chamber 13, at least the second space located above the filter 16 mentioned below.
  • the microfluidic component 1 includes a filter 16 arranged in said chamber 13 and separating said chamber 13 into two spaces.
  • the two spaces are for example superimposed and thus designated lower space 130 located under the filter and upper space 131 located above the filter and under the membrane 18.
  • This filter 16 is preferably produced in whole or in part in the form of a film flexible and thin, held in the space formed by the chamber so as to allow passage from one space to another only through the pores of the filter 16.
  • the film advantageously has an elastic deformability allowing it to stretch during of the exertion of a bearing force in a substantially vertical direction, this elastic deformability having a sufficient level to reach the lower surface of the chamber 13.
  • the filter 16 has an average pore diameter of between 0.2 ⁇ m and 50 ⁇ m , for example between 0.2 ⁇ m and 1 ⁇ m for the separation of microorganisms.
  • the diameter of the pores is of course adapted to ensure separation between different biological species present in the sample.
  • the filter 16 will for example be composed of a film of thickness, dimensions and constitution adapted to deform to the bottom of the chamber 13 with respect to its anchor points.
  • the filter may also be made of a transparent material, for example with the same transparency characteristics as the membrane.
  • the filter may have a pore diameter ranging from 0.2 to 2 ⁇ m to retain the bacteria.
  • the micro-fluidic component can advantageously comprise a rough bearing surface 17 arranged on the bottom of the chamber 13.
  • This rough bearing surface 17 extends over a major part of the bottom of the chamber. It has an average surface roughness parameter of between 0.01 ⁇ m and 10 ⁇ m, preferably between 0.2 ⁇ m and 3 ⁇ m.
  • This rough bearing surface 17 is intended to allow mechanical lysis of the biological species present in the biological sample placed in the device.
  • the mechanical lysis is carried out by grinding said biological species, by abrasion on said rough support surface.
  • the grinding operation is implemented by a friction movement of the biological species against the rough bearing surface, using a suitable grinding device.
  • This member will for example be a spatula or a rod, for example made of plastic or metallic material.
  • This member is applied from the outside of the chamber 13 and its end is applied against the outer surface of the membrane 18 so as to stretch the membrane 18 and the filter towards the bottom of the chamber and thus rub the biological species present in a sample against the rough bearing surface 17.
  • the casing can advantageously incorporate means for heating the internal space of the chamber, composed for example of at least one heating resistor.
  • the resistance is for example fixed under the lower wall of the housing.
  • a power source will for example be provided to power the resistor.
  • the power source will include for example one or more electric batteries, providing enough energy to heat the chamber to a temperature within the range defined above, i.e. from 20°C to 100°C .
  • other heating means could be employed, comprising for example a conductive ink deposited by printing or screen printing under the lower wall of the casing.
  • the lower wall of the component and the membrane can be made of transparent materials, in particular to implement detection by biomolecular amplification.
  • This component makes it possible in particular to inject the sample in flow and to treat it in flow.
  • in flow is meant that the sample is injected in liquid form into the component continuously, via its first channel 14, without leaving the sample from the component.
  • the treatment solutions applied to it these being injected in fluid form continuously into the component, via its first channel 14, without having to remove the sample present in the component.
  • the filter 16 the target biological species are maintained in the lower space 130 of the chamber 13 of the component.
  • flow is opposed to the term “reflux”, which consists for its part of injecting a fluid via the second channel 15 of the component, to drive the sample out of the component, via the first channel 14.
  • the method can be implemented according to the two variants represented on the figure 1 and 2 .
  • microfluidic component has the particular advantage of optimizing the quantity of target biological species obtained and of concentrating them as much as possible in the lower space 130 of the chamber 13 of the component 1.
  • a sample P is first taken.
  • This sample may consist, for example, of leaves of the targeted plant compound or of any other sample for which the method of the invention may prove to be suitable. They may in particular be cells of a living being or any other substance comprising DNA molecules and capable of presenting biological species in an active form and in a dormant form.
  • E0 This sample is initially processed in order to obtain an ECH sample that can be used and easily separated into two parts A, B of equal quantities.
  • This initial treatment may consist of grinding the sample and then carrying out an extraction in order to obtain a liquid ready for analysis.
  • This type of processing is well known and is not detailed in the present application.
  • the ECH sample obtained, in a liquid form, can then be the subject of the method of the invention.
  • the method of the invention can be implemented by using the same microfluidic component 1 or by using two identical microfluidic components in parallel.
  • the two parts A, B of the ECH sample are injected into two identical components in parallel and the steps of the method are carried out simultaneously in the two components to analyze the two parts of the sample.
  • two identical micro-fluidic components are used in parallel, to analyze separately a first part of the sample and a second part of the sample.
  • E1 The ECH sample, containing the target biological species, is divided into two parts A, B of equal quantities.
  • the first part A of the treated sample is injected in flow into the first microfluidic component through its first channel 14.
  • the biological species present in this first part of the sample remain trapped in the lower space 130 of the chamber 13 of the micro-fluidic component, thus allowing their concentration.
  • the biological species present in this second part of the sample remain trapped in the lower space 130 of the chamber 13 of the microfluidic component, thus allowing their concentration.
  • E4 The first part A of the sample undergoes no treatment or at most a mild treatment T1.
  • This mild treatment T1 can consist of a simple washing with a buffer, for example an aqueous buffer with mild surfactants (0.01% Tween 20 buffer - registered trademark). This gentle T1 treatment should not destroy the target biological species.
  • the buffer is injected in flow through the first channel 14 of the component.
  • This active T2 treatment makes it possible to break the DNA molecules of the active F2 form of the biological species.
  • the F2′ DNA molecules broken during the active treatment step are not retained by the filter 16 of the component during the purification step. They are therefore evacuated by elution by passing through the filter 16 then through the second channel 15 of the microfluidic component.
  • the remaining biological species are then advantageously purified by injecting a buffer liquid through the first channel of the component, this liquid passes through the chamber and the filter 16 and is then evacuated via the second channel 15.
  • the active treatment T2 is chosen to be suitable for destroying the active form F2 of the targeted biological species in the ECH sample and for maintaining the dormant form F1 of the targeted biological species. Depending on the biological species and its active form, the most effective active treatment possible is therefore chosen.
  • the active treatment comprises at least one chemical or biochemical treatment of the second part of the sample, carried out in flow through the component in which the biological species F1, F2 have been trapped during the previous step E3.
  • Each active treatment applied can make it possible to gradually eliminate the active form F2 of the targeted biological species.
  • the substance is injected in flow into component 1, through its first channel 14, and thus comes directly to encounter the biological species F1, F2 present in the lower space 130 of chamber 13 of component 1.
  • E8/E9 This is the step for processing the results obtained. It is in fact a question of comparing the quantity Q1 of biological species obtained in the first part of the sample and the quantity Q2 of biological species obtained in the second part of the sample.
  • microfluidic components are also used in parallel.
  • the gentle treatment T1 and the active treatment T2 carried out respectively on the first part A of the sample and on the second part B of the sample can be implemented beforehand before the injection of the samples into each of the two components.
  • micro-fluidics used in parallel steps E20 and E30 on the picture 2 ).
  • the active treatment T2 can then be continued in flux to eliminate the active forms F2 of the biological species which could still be present, after the injection of the second part B of the sample into the component.
  • Steps E4 and E5 described above, relating to the injection in flow of the two parts A, B of the sample into each of the two components remain identical to those described above.
  • the active treatment T2 having been undertaken during step E30 on the second part B of the sample, the DNA molecules F2' are not retained by the filter 16 of the component and are evacuated from component 1.
  • Steps E40 and E50 consist in processing in flow each of the samples trapped during steps E4 and E5 (gentle treatment T1 possible in step E40 for the first part A of the sample and active treatment T2 in flow in step E50 for the second part B of the sample).
  • the active treatment T2 makes it possible to evacuate from component 1 the residual active forms, at the level of the second part B of the sample.
  • the active treatment comprises at least one chemical or biochemical treatment as described above for the first embodiment variant.
  • Steps E6, E7, E8 and E9 described above for the first embodiment variant remain identical.
  • Bs Bacillus subtillis
  • sample no. 1 In sample no. 1, it is therefore observed that the bacteria have been broken by the heat treatment, the DNA molecules have not been retained in the microfluidic component, which explains why the detection is negative. It is concluded that sample no. 1 of 10,000 Bs bacteria/500 ⁇ L consists solely of the biological species Bs in its vegetative (active) form.

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EP22182863.5A 2021-07-07 2022-07-04 Verfahren zur analyse einer probe zum nachweis einer aktiven form einer biologischen spezies Pending EP4116429A1 (de)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
FR2107363A FR3125124A1 (fr) 2021-07-07 2021-07-07 Procédé d'analyse d'un échantillon pour y détecter la présence d'une forme active d'une espèce biologique

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EP4116429A1 true EP4116429A1 (de) 2023-01-11

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EP22182863.5A Pending EP4116429A1 (de) 2021-07-07 2022-07-04 Verfahren zur analyse einer probe zum nachweis einer aktiven form einer biologischen spezies

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